《英语教学》（英文版）Working Safely with Radioactive Materials

Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-47 (Electronic) 6 Working Safely with Radioactive materials William R.. Volny r. Licensing and Certification 143 Do You need a license to handle radioactive Materials? 143 Who Do You Contact to Begin the Process of Becoming Licensed or Certified to Use Radioactivity? Selecting and Ordering a Radioisotope 144 Which Radiochemical Is Most Appropriate for Your Research 144 What Quantity of Radioactivity Should You Purchase? When Should You order the material? How Do You Calculate the Amount of Remaining How Long after the Reference Date Can You Use Your material? 149 Can You Compensate by Adding More Radiochemical If the Reference Date Has Long Passed? Handling Radioactive Shipments What Should You Do with the Radioactive Shipment A Wipe Test Detected Radioactivity on the Outside When lt arrives? 50 of the vial. Does this indicate a problem? 5 14l

141 6 Working Safely with Radioactive Materials William R. J. Volny Jr. Licensing and Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Do You Need a License to Handle Radioactive Materials? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Who Do You Contact to Begin the Process of Becoming Licensed or Certified to Use Radioactivity? . . . . . . . . . . . 144 Selecting and Ordering a Radioisotope . . . . . . . . . . . . . . . . . . . 144 Which Radiochemical Is Most Appropriate for Your Research? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 What Quantity of Radioactivity Should You Purchase? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 When Should You Order the Material? . . . . . . . . . . . . . . . . . 148 How Do You Calculate the Amount of Remaining Radiolabel? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 How Long after the Reference Date Can You Use Your Material? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Can You Compensate by Adding More Radiochemical If the Reference Date Has Long Passed? . . . . . . . . . . . . . . 150 Handling Radioactive Shipments . . . . . . . . . . . . . . . . . . . . . . . . . 150 What Should You Do with the Radioactive Shipment When It Arrives? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 A Wipe Test Detected Radioactivity on the Outside of the Vial. Does This Indicate a Problem? . . . . . . . . . . . . 151 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)

Your count -Rate meter detects radiation on the Outside of a Box Containing I mCi of aP Labeled dATP Is It Contaminated? 52 You Received 250 u Ci of P and the Box Wasn't Labeled Radioactive. Isnt This a Dangerous Mistake? esigning Your Experiments 53 How Do You Determine the Molarity and Mass in the vial of material? 53 How Do You Quantitate the Amount of Radioactivity for Your reaction? 155 Storing Radioactive Materials 56 What Causes the Degradation of a Radiochemical? 156 What Can You do to maximize the lifetime and Potency of a radiochemical? 156 What Is the Stability of a Radiolabeled Protein or Nucleic Acid 57 Radioactive Waste: What Are Your Options and obligatio Handling Radioactivity: Achieving Minimum Dose 159 How Is Radioactive Exposure Quantified and What Are the allowable doses? 59 Monitoring Technology: What's the Difference between a Count -Rate Counter and a dose-Rate Meter? 59 What Are the Elements of a Good Overall Monitoring 161 Strategy What Can You do to achieve minimum radioactive Dose? 162 How Can You Organize Your Work Area to Minimize Your Exposure to Radioactivity? How Can You concentrate a radioactive solution? Bibliography 166 Appendix A: Physical Properties of Common Radionuclides The information within this chapter is designed as a supple. ment, not a replacement, to the training provided by your institu tional rules and/or radiation safety officer. At the very least, there are some 10 fundamental rules to consider when working with radioactivity(Amerhsam International, 1974) 1. Understand the nature of the hazard and get practical ahead to minimize time spent handling radioactivity

Your Count-Rate Meter Detects Radiation on the Outside of a Box Containing 1mCi of a 32P Labeled dATP. Is It Contaminated? . . . . . . . . . . . . . . . . . . . . . . . . . . 152 You Received 250mCi of 32P and the Box Wasn’t Labeled Radioactive. Isn’t This a Dangerous Mistake? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Designing Your Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 How Do You Determine the Molarity and Mass in the Vial of Material? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 How Do You Quantitate the Amount of Radioactivity for Your Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Storing Radioactive Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 What Causes the Degradation of a Radiochemical? . . . . . . 156 What Can You Do to Maximize the Lifetime and Potency of a Radiochemical? . . . . . . . . . . . . . . . . . . . . . . . . 156 What Is the Stability of a Radiolabeled Protein or Nucleic Acid? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Radioactive Waste: What Are Your Options and Obligations? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Handling Radioactivity: Achieving Minimum Dose . . . . . . . . . . 159 How Is Radioactive Exposure Quantified and What Are the Allowable Doses? . . . . . . . . . . . . . . . . . . . . . 159 Monitoring Technology: What’s the Difference between a Count-Rate Counter and a Dose-Rate Meter? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 What Are the Elements of a Good Overall Monitoring Strategy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 What Can You Do to Achieve Minimum Radioactive Dose? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 How Can You Organize Your Work Area to Minimize Your Exposure to Radioactivity? . . . . . . . . . . . . . . . . . . . . . 164 How Can You Concentrate a Radioactive Solution? . . . . . . 164 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Appendix A: Physical Properties of Common Radionuclides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 The information within this chapter is designed as a supple￾ment, not a replacement, to the training provided by your institu￾tional rules and/or radiation safety officer. At the very least, there are some 10 fundamental rules to consider when working with radioactivity (Amerhsam International, 1974): 1. Understand the nature of the hazard, and get practical training. 2. Plan ahead to minimize time spent handling radioactivity. 142 Volny Jr

3. Distance yourself appropriately from sources of radiation 4. Use appropriate shielding for the radiation. 5. Contain radioactive materials in defined work areas. 6. Wear appropriate protective clothing and dosimeters. 7. Monitor the work area frequently for contamination ontrol 8. Follow the local rules and safe ways of working 9. Minimize accumulation of waste and dispose of it by appropriate routes. 10. After completion of work, monitor yourself; then wash and monitor again LICENSING AND CERTIFICATION Do You need a license to handle radioactive materials? Q Whichever type of license is granted by the Nuclear Regulatory ommission (NRC), it tends to be a single license issued to the institution itself, to regulate its entire radioisotope usage. Separate licenses are not normally granted to the various departments or to individuals at that institution. However, everyone who works with radioactive materials at a licensed institution must be trained nd approved to use the radioactive materials. Keep in mind that some states may have control over radioactive materials not con- trolled by the NrC. In addition, in"agreement states, the NRc requirements are regulated and controlled by a state agency Universities, governmental institutions, or industry are usually licensed to use radionuclides under a Type A License of Broad Scope (U.s. Nuclear Regulatory Commission Regulatory Guide 1980). This is the most comprehensive license available to an insti tution. It requires that the institution have a radiation safety com ted radiation safety officer(Rso), and detailed radiation protection and training procedures. Researchers who want to use radionuclides in their work must present the proposal to the radiation safety committee and have it approved before being able to carry out the experiments. There are other types of licenses issued by nRC or by agree ment states. For example, these may be specific by-product material licenses of limited scope, specific licenses of broad scope licenses for source or special nuclear materials, or licenses for kilocurie irradiation sources (U.S. Nuclear Regulatory Commis sion Regulatory Guide, 1979, 1976). By-product materials are the radionuclides that form during reactor processes. The most commonly used radionuclides, P, P, S, H,C, and I are all -product materials. The licensing of by-product material is Working Safely with Radioactive Materials 143

3. Distance yourself appropriately from sources of radiation. 4. Use appropriate shielding for the radiation. 5. Contain radioactive materials in defined work areas. 6. Wear appropriate protective clothing and dosimeters. 7. Monitor the work area frequently for contamination control. 8. Follow the local rules and safe ways of working. 9. Minimize accumulation of waste and dispose of it by appropriate routes. 10. After completion of work, monitor yourself; then wash and monitor again. LICENSING AND CERTIFICATION Do You Need a License to Handle Radioactive Materials? Whichever type of license is granted by the Nuclear Regulatory Commission (NRC), it tends to be a single license issued to the institution itself, to regulate its entire radioisotope usage. Separate licenses are not normally granted to the various departments or to individuals at that institution. However, everyone who works with radioactive materials at a licensed institution must be trained and approved to use the radioactive materials. Keep in mind that some states may have control over radioactive materials not con￾trolled by the NRC. In addition, in “agreement states,” the NRC requirements are regulated and controlled by a state agency. Universities, governmental institutions, or industry are usually licensed to use radionuclides under a Type A License of Broad Scope (U.S. Nuclear Regulatory Commission Regulatory Guide, 1980).This is the most comprehensive license available to an insti￾tution. It requires that the institution have a radiation safety com￾mittee, an appointed radiation safety officer (RSO), and detailed radiation protection and training procedures. Researchers who want to use radionuclides in their work must present the proposal to the radiation safety committee and have it approved before being able to carry out the experiments. There are other types of licenses issued by NRC or by agree￾ment states. For example, these may be specific by-product material licenses of limited scope, specific licenses of broad scope, licenses for source or special nuclear materials, or licenses for kilocurie irradiation sources (U.S. Nuclear Regulatory Commis￾sion Regulatory Guide, 1979, 1976). By-product materials are the radionuclides that form during reactor processes. The most commonly used radionuclides, 32P, 33P, 35S, 3 H, 14C, and 125I are all by-product materials. The licensing of by-product material is Working Safely with Radioactive Materials 143

covered in detail under Title 10, Code of Federal Regulations (CFR), Part 30, Rules of General Applicability to Licensing of Byproduct Material (10CFR Part 30), and 10CFR Part 33, Spe- cific Domestic Licenses of Broad Scope for Byproduct Material (10CFR Part 33) For more information, a recent publication by the NRC is now available entitled: Consolidated guidance about materials Licenses. Program-Specific Guidance about Academic Research and Development, and other Licenses of Limited Scope. Final Report U.S. Regulatory Commission, Office of Nuclear Material Safety and Safeguards. NOREG-1556, Vol 7. M. L. Fuller, R.P. Hayes, A S Lodhi, G. W. Purdy, December 1999. You can also find informationontheNrcWebsitewww.Nrc.gov.TheAtomic Energy Control Board, or AECB, governs radioactive use in CanadaTheirWebsiteiswww.aecb-ccea.gc.ca Who Do You Contact to Begin the Process of Becoming Licensed or Certified to Use Radioactivity? If you want to use radioactivity in your research, you may need o become an authorized user at your institution. First, decide what type of isotope or isotopes will be used in your research, the application, how much material you will need, disposal methods, and for how long you will use it. Then, present this information to your radiation safety officer or radiation safety committee so that hey can determine whether such radionuclide use is possible under your institution's license. If the request is approved, carry out the requirements stated on your institution's license to become an authorized user operating in an approved laboratory SELECTING AND ORDERING A RADIOISOTOPE Which Radiochemical Is Most Appropriate for Your research? The Institution's Perspective Your institution's license defines specific limits to the type and amount of radionuclide allowable on site(this includes on-site aste). Before determining how much material you think you'll need, find out how much you' ll be allowed to have in your lab at any one time. You can then get an idea about how or if you'll need to space out the work requiring radioactivity Your perspective These are some of the most important parameters to consider when deciding which isotope to use

covered in detail under Title 10, Code of Federal Regulations (CFR), Part 30, Rules of General Applicability to Licensing of Byproduct Material (10CFR Part 30), and 10CFR Part 33, Spe￾cific Domestic Licenses of Broad Scope for Byproduct Material (10CFR Part 33). For more information, a recent publication by the NRC is now available entitled: Consolidated Guidance about Materials Licenses. Program-Specific Guidance about Academic Research and Development, and other Licenses of Limited Scope. Final Report U.S. Regulatory Commission, Office of Nuclear Material Safety and Safeguards. NOREG-1556, Vol. 7. M. L. Fuller, R. P. Hayes, A. S. Lodhi, G. W. Purdy, December 1999. You can also find information on the NRC Web site www.NRC.gov. The Atomic Energy Control Board, or AECB, governs radioactive use in Canada. Their Web site is www.aecb-ccea.gc.ca. Who Do You Contact to Begin the Process of Becoming Licensed or Certified to Use Radioactivity? If you want to use radioactivity in your research, you may need to become an authorized user at your institution. First, decide what type of isotope or isotopes will be used in your research, the application, how much material you will need, disposal methods, and for how long you will use it. Then, present this information to your radiation safety officer or radiation safety committee so that they can determine whether such radionuclide use is possible under your institution’s license. If the request is approved, carry out the requirements stated on your institution’s license to become an authorized user operating in an approved laboratory. SELECTING AND ORDERING A RADIOISOTOPE Which Radiochemical Is Most Appropriate for Your Research? The Institution’s Perspective Your institution’s license defines specific limits to the type and amount of radionuclide allowable on site (this includes on-site waste). Before determining how much material you think you’ll need, find out how much you’ll be allowed to have in your lab at any one time.You can then get an idea about how or if you’ll need to space out the work requiring radioactivity. Your Perspective These are some of the most important parameters to consider when deciding which isotope to use. 144 Volny Jr

●o● specific activity diluted o molecules of compound of compound Figure 6.1 Diagrammatical representation of radiochemicals at low and high specific ctivity, and at high specific activity in a diluent. From Guide to the Self-decomposition of Radiochemicals, Amersham International, plc, 1992, Buckinghamshire, U.K. Reprinted by permission of Amersham Pharmacia Biotech Radionuclide, Energy, and Type of Emission(Alpha, Beta, Gamma, X ray, etc In most cases you won't have the choice. You will choose the adionuclide because of its elemental properties, and its reactivity in reference to the experiment, not its type of emission. Each radionuclide has its unique emission spectrum. The spectra are important in determining how you detect the radioactivity in your samples. This is discussed more fully later in the chapter. Specific Activity and Radioactive Concentration The highest specific activity and the highest radioactive con centration tend to be the best since it means that there will be the greatest number of radioactive molecules in a given mass and volume(Figure 6.1). But there are two caveats to this ideal. The first is that as you increase the specific activity, you decrease the molar concentration of your desired molecule. This molecule will become the limiting reagent and possibly slow down or halt the reaction. The second danger is that at high specific activities and/or radioactive concentrations, the rate of radiolytic decomposition will increase. These parameters are discussed in more detail in Chapter 14, Nucleic Acid Hybridization o take an example, a standard random priming labeling reaction requires 50 uCi (1.85 MBq)* of P dNTP(Feinberg *In the United States the unit of activity of"Curie"is still used The unit of common usage is the Becquerel(Bq). Whereas I Curie=3. 7 x 10 disintegrations per second(dps), the Bq=1 dps. For example, to convert picocuries(10 Curies)to Working Safely with Radioactive Materials 145

Radionuclide, Energy, and Type of Emission (Alpha, Beta, Gamma, X ray, etc.) In most cases you won’t have the choice. You will choose the radionuclide because of its elemental properties, and its reactivity in reference to the experiment, not its type of emission. Each radionuclide has its unique emission spectrum. The spectra are important in determining how you detect the radioactivity in your samples. This is discussed more fully later in the chapter. Specific Activity and Radioactive Concentration The highest specific activity and the highest radioactive con￾centration tend to be the best since it means that there will be the greatest number of radioactive molecules in a given mass and volume (Figure 6.1). But there are two caveats to this ideal. The first is that as you increase the specific activity, you decrease the molar concentration of your desired molecule. This molecule will become the limiting reagent and possibly slow down or halt the reaction.The second danger is that at high specific activities and/or radioactive concentrations, the rate of radiolytic decomposition will increase. These parameters are discussed in more detail in Chapter 14, “Nucleic Acid Hybridization.” To take an example, a standard random priming labeling reaction requires 50mCi (1.85MBq)* of 32P dNTP (Feinberg Working Safely with Radioactive Materials 145 Figure 6.1 Diagrammatical representation of radiochemicals at low and high specific activity, and at high specific activity in a diluent. From Guide to the Self-decomposition of Radiochemicals, Amersham International, plc, 1992, Buckinghamshire, U.K. Reprinted by permission of Amersham Pharmacia Biotech. *In the United States the unit of activity of “Curie” is still used. The unit of common usage is the Becquerel (Bq). Whereas 1 Curie = 3.7 ¥ 1010 disintegrations per second (dps), the Bq = 1 dps. For example, to convert picocuries (10-12 Curies) to

and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50uCi translates to 16.6 femtomoles of P dNTP being added to the reaction mix, while 50 uCi of aP labeled dNTP at a specific activity of 6000 Ci/mmol will add only 8.3 femtomoles to the reac tion. Unless sufficient unlabeled dntP is added the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME(absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it,'s worthwhile to consider the following impacts of the label location: First, will the labels location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label's presence in a nucleic acid probe interfere with the probe's ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, Nucleic Acid Hybridization. There are some reactions where the location of label is not criti- cal. a thymidine uptake assay is one such case. The labeling will york just as effectively whether the tritium is on the methyl group ng. The Form and Quantity of the radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a com- patible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be Becquerels, divide by 27(27.027): 50 uCi=50 x 10-Ci=(50 10°Ci×3.7×l00 dps/Ci)=1.85×10°dps=1.85×10°Bq= 185MBq.) 146

and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50mCi translates to 16.6 femtomoles of 32P dNTP being added to the reaction mix, while 50mCi of a 32P labeled dNTP at a specific activity of 6000Ci/mmol will add only 8.3 femtomoles to the reac￾tion. Unless sufficient unlabeled dNTP is added, the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol material. Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME (absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it’s worthwhile to consider the following impacts of the label location: First, will the label’s location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label’s presence in a nucleic acid probe interfere with the probe’s ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, “Nucleic Acid Hybridization.” There are some reactions where the location of label is not criti￾cal. A thymidine uptake assay is one such case. The labeling will work just as effectively whether the tritium is on the methyl group or on the ring. The Form and Quantity of the Radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a com￾patible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be 146 Volny Jr. Becquerels, divide by 27 (27.027): 50mCi = 50 ¥ 10-6Ci = (50 ¥ 10-6Ci ¥ 3.7 ¥ 1010dps/Ci) = 1.85 ¥ 106dps = 1.85 ¥ 106Bq = 1.85MBq.)

the best solvent for long-term storage. From a manufacturing per spective, the radiochemical is supplied in a solvent that is a com promise between the stability and solubility of the compound and the investigators convenience. Some common solvents to consider, and the reasons they are used Ethanol, 2%. Added to aqueous solvents where it acts as free radical scavenger and will extend the shelf life of the radio- labeled compound Toluene or benzene. Most often used to increase stability of the radiolabeled compound, and increase solubility of nonpolar ds, such as lipids B-mercaptoethanoL, 5mM. Helps to minimize the release of radioactive sulfur from amino acids and nucleotides in the form of sulfoxides and other volatile molecules Colored dyes Added for the investigator's convenience to visualize the presence of the radioactivity When not in use. the "stock" solution of the radioactive compound is capped and usually refrigerated to minimize olatilization/evaporation What Quantity of Radioactivity Should You Purchase? There are three things to consider when deciding how much aterial to purchase 1. How much activity (radioactivity) will be used and over what period? 2. What are the institutional limits affecting the amounts of radioisotope chosen that your lab may be authorized to 3. What are the decomposition rate of your radiolabeled compound and its half-life. n general you will want to purchase as large a quantity as possible to save on initial cost, while at the same time not com promising the quality of the results of the research by using decomposed material. For example, certain forms of tritiated hymidine can have radiolytic decomposition rates(thymidine degradation) of 4% per week. This decomposition rate is not to be confused with tritium's decay rate, or half-life, which is over 12 years. Stocking up on such rapidly decomposing material, or by using it for more than just a few months could compromise experiments carried out later in the products life. Working Safely with Radioactive Materials 147

the best solvent for long-term storage. From a manufacturing per￾spective, the radiochemical is supplied in a solvent that is a com￾promise between the stability and solubility of the compound and the investigator’s convenience. Some common solvents to consider, and the reasons they are used: • Ethanol, 2%. Added to aqueous solvents where it acts as a free radical scavenger and will extend the shelf life of the radio￾labeled compound. • Toluene or benzene. Most often used to increase stability of the radiolabeled compound, and increase solubility of nonpolar compounds, such as lipids. • 2-mercaptoethanol, 5 mM. Helps to minimize the release of radioactive sulfur from amino acids and nucleotides in the form of sulfoxides and other volatile molecules. • Colored dyes. Added for the investigator’s convenience to visualize the presence of the radioactivity. When not in use, the “stock” solution of the radioactive compound is capped and usually refrigerated to minimize volatilization/evaporation. What Quantity of Radioactivity Should You Purchase? There are three things to consider when deciding how much material to purchase: 1. How much activity (radioactivity) will be used and over what period? 2. What are the institutional limits affecting the amounts of radioisotope chosen that your lab may be authorized to use? 3. What are the decomposition rate of your radiolabeled compound and its half-life. In general you will want to purchase as large a quantity as possible to save on initial cost, while at the same time not com￾promising the quality of the results of the research by using decomposed material. For example, certain forms of tritiated thymidine can have radiolytic decomposition rates (thymidine degradation) of 4% per week. This decomposition rate is not to be confused with tritium’s decay rate, or half-life, which is over 12 years. Stocking up on such rapidly decomposing material, or by using it for more than just a few months could compromise experiments carried out later in the product’s life. Working Safely with Radioactive Materials 147

When Should You order the materia? Analvsis Date Ideally you will want to schedule your experiments and your adiochemical shipments such that the material arrives at its maximum level of activity and lowest level of decomposition. This will tend to be when the product is newer, or nearer its analy date(the date on which the compound passes quality control tests and is diluted appropriately so that the radioactive concentration and specific activity will be as those stated on the reference date) Some isotopes and radiochemicals decompose slowly, so it is not always necessary to take this suggestion to the extreme. As you use a radiolabeled product, you' ll come to know how long you can use it in your work. AnI labeled ligand will not last as long as aC labeled sugar. An inorganic radiolabeled compound, such as Na I or sodium chromate, will decompose at the isotopes rate of decay, whereas a labeled organic compound, such as the tritiated thymidine alluded to earlier, will decompose at a much faster rate than the half-life of the isotope would indicate Manufacturers take this into account by having a terminal sale date The material will only be sold for so long before it is removed from its stores. Up until this date you will be able to purchase the material and still expect to use it over a reasonable period of time Reference Date The reference date is the day on which you will have the stated amount of material. If you purchased a 1 mCi vial of 3 P dCTP, yo will have greater than l mCi (37 MBq) prior to the reference date, l mCi on that date, and successively less beyond the reference date. (Note that since you will most likely receive your radioac tive material prior to reference, it is possible to exceed possession limits; consider this when determining limits on your radiation license ) In the case of longer-lived radioisotopes, such as H and C, the analysis date will also serve as the reference date How Do you calculate the amount of emaining Radiolabel? is to use the following exponential decay equation. For conve nience's sake, most manufacturers of radiochemicals provide decay charts in their catalogs for commonly used isotopes. This equation comes in handy for the less common isotopes. A= Aoe 0.693tT 148

When Should You Order the Material? Analysis Date Ideally you will want to schedule your experiments and your radiochemical shipments such that the material arrives at its maximum level of activity and lowest level of decomposition. This will tend to be when the product is newer, or nearer its analysis date (the date on which the compound passes quality control tests and is diluted appropriately so that the radioactive concentration and specific activity will be as those stated on the reference date). Some isotopes and radiochemicals decompose slowly, so it is not always necessary to take this suggestion to the extreme. As you use a radiolabeled product, you’ll come to know how long you can use it in your work. An 125I labeled ligand will not last as long as a 14C labeled sugar. An inorganic radiolabeled compound, such as Na125I or sodium 51chromate, will decompose at the isotope’s rate of decay, whereas a labeled organic compound, such as the tritiated thymidine alluded to earlier, will decompose at a much faster rate than the half-life of the isotope would indicate. Manufacturers take this into account by having a terminal sale date.The material will only be sold for so long before it is removed from its stores. Up until this date you will be able to purchase the material and still expect to use it over a reasonable period of time. Reference Date The reference date is the day on which you will have the stated amount of material. If you purchased a 1 mCi vial of 32P dCTP, you will have greater than 1 mCi (37MBq) prior to the reference date, 1mCi on that date, and successively less beyond the reference date. (Note that since you will most likely receive your radioac￾tive material prior to reference, it is possible to exceed possession limits; consider this when determining limits on your radiation license.) In the case of longer-lived radioisotopes, such as 3 H and 14C, the analysis date will also serve as the reference date. How Do You Calculate the Amount of Remaining Radiolabel? The most straightforward way of calculating radioactive decay is to use the following exponential decay equation. For conve￾nience’s sake, most manufacturers of radiochemicals provide decay charts in their catalogs for commonly used isotopes. This equation comes in handy for the less common isotopes. A = A0e-0.693t/T 148 Volny Jr

where Ao is the radioactivity at reference date t is the time between reference date and the time you are cal culating for, Tis the half-life of the isotope(note that both t and T must have the same units of time) It is easy to use the aforementioned decay charts as shown in the following two examples. Say you had 250uCi of>>s methionine at a certain reference date and the radioactive concentration was 15mCi/ml. Now it is 25 days after that reference date You calculate your new radioac tive concentration and total activity in the vial by looking on the chart to locate the fraction under the column and row that corre- sponds to 25 days postreference. This number should be 0.820 Multiply your starting radioactive concentration by this fraction to obtain the new radioactive concentration: 15mCi/ml x0.820=12.3 mCi/ml The total amount of activity can be likewise calculated for S with a half-life of 87 4 days; namely A Age- 15exp(0.693×25/874)=123mCi/m For the second example you can find out how much activity you had before the reference date. Some decay charts only have postreference fractions, but if you have a l mCi vial of P dUTP at 10mCi/ml, and it is 5 days prior to the reference date, how do you figure out how much you have? Go to the column and row on the P decay chart corresponding to 5 days postreference There you will see the fraction 0.872. You will divide your ref- erence activity and radioactive concentration by this number to obtain the proper amount of activity present, or 1/0.872 1.15 mCi. Note that the values should be greater than the stated amounts of activity and the referenced radioactive concentration For the calculation method you are now looking for Ao. Therefore Ao= Aeos=10exp(0.693 x 5//25)=11.5mCi/ml, using a half-life of 25 days for> P How Long after the Reference Date Can You Use Your material? Radioactively labeled compounds do not suddenly go bad after the reference date. It isnt an expiration date. It is used as a benchmark by which you can anchor your decay calculations Working Safely with Radioactive Materials 149

where A0 is the radioactivity at reference date, t is the time between reference date and the time you are cal￾culating for, T is the half-life of the isotope (note that both t and T must have the same units of time). It is easy to use the aforementioned decay charts as shown in the following two examples. Say you had 250mCi of 35S methionine at a certain reference date, and the radioactive concentration was 15mCi/ml. Now it is 25 days after that reference date. You calculate your new radioac￾tive concentration and total activity in the vial by looking on the chart to locate the fraction under the column and row that corre￾sponds to 25 days postreference. This number should be 0.820. Multiply your starting radioactive concentration by this fraction to obtain the new radioactive concentration: 15 mCi/ml ¥ 0.820 = 12.3 mCi/ml The total amount of activity can be likewise calculated for 35S with a half-life of 87.4 days; namely A = A0e-0.693t/T = 15 exp(-0.693 ¥ 25/87.4) = 12.3mCi/ml. For the second example you can find out how much activity you had before the reference date. Some decay charts only have postreference fractions, but if you have a 1 mCi vial of 33P dUTP at 10 mCi/ml, and it is 5 days prior to the reference date, how do you figure out how much you have? Go to the column and row on the 33P decay chart corresponding to 5 days postreference. There you will see the fraction 0.872. You will divide your ref￾erence activity and radioactive concentration by this number to obtain the proper amount of activity present, or 1/0.872 = 1.15 mCi. Note that the values should be greater than the stated amounts of activity and the referenced radioactive concentration. For the calculation method you are now looking for A0. Therefore A0 = Ae0.693t/T = 10exp(0.693 ¥ 5/25) = 11.5mCi/ml, using a half-life of 25 days for 33P. How Long after the Reference Date Can You Use Your Material? Radioactively labeled compounds do not suddenly go bad after the reference date. It isn’t an expiration date. It is used as a benchmark by which you can anchor your decay calculations as described above. Working Safely with Radioactive Materials 149

Table 6.1 Shelf Lives for Commonly Used Only you can determine how long you can use your radio- isotope after the reference date. The answer depends on the isotope, the compound it's bound to, the experiment, storage, the formulation of the product, and the like. Table 6.1 lists the genera ranges for the most commonly used radioisotopes, which is guideline only. As you carry out your work, you will discover when your material starts to give poorer results Can You Compensate by Adding More Radiochemical If the Reference Date Has Long Pass Sometimes it is not that simple. As an example of the complex- ities involved with radiolytic decomposition, suppose you had a vial of 32P gamma labeled ATP that you routinely use to label the 5 end of dna via T4 Polynucleotide kinase. If one half-life has passed since the reference date(14.28 days), you will have 50% of the stated radioactivity remaining. You might still achieve satisfactory 5" end labeling with T4 Polynucleotide kinase if you double the amount of theP added to the reaction. Often however, you may find that though you have compensated for the adioactive decay by adding more material, you have also intro- duced more of the decomposition products, which will be frag ments of the original labeled compound and free radicals. You also will have added more of the solute that might be present in the stock vial. These contaminants and decomposition products ca significantly interfere with the reaction mechanism and compro mise your results HANDLING RADIOACTIVE SHIPMENTS What should You Do with the Radioactive Shipment When it Ar The radiation safety officer is responsible for ensuring that radioactive materials are received in satisfactory condition, but

Only you can determine how long you can use your radio￾isotope after the reference date. The answer depends on the isotope, the compound it’s bound to, the experiment, storage, the formulation of the product, and the like. Table 6.1 lists the general ranges for the most commonly used radioisotopes, which is a guideline only.As you carry out your work, you will discover when your material starts to give poorer results. Can You Compensate by Adding More Radiochemical If the Reference Date Has Long Passed? Sometimes it is not that simple. As an example of the complex￾ities involved with radiolytic decomposition, suppose you had a vial of 32P gamma labeled ATP that you routinely use to label the 5¢ end of DNA via T4 Polynucleotide kinase. If one half-life has passed since the reference date (14.28 days), you will have 50% of the stated radioactivity remaining. You might still achieve satisfactory 5¢ end labeling with T4 Polynucleotide kinase if you double the amount of the 32P added to the reaction. Often, however, you may find that though you have compensated for the radioactive decay by adding more material, you have also intro￾duced more of the decomposition products, which will be frag￾ments of the original labeled compound and free radicals.You also will have added more of the solute that might be present in the stock vial. These contaminants and decomposition products can significantly interfere with the reaction mechanism and compro￾mise your results. HANDLING RADIOACTIVE SHIPMENTS What Should You Do with the Radioactive Shipment When It Arrives? The radiation safety officer is responsible for ensuring that radioactive materials are received in satisfactory condition, but 150 Volny Jr. Table 6.1 Shelf Lives for Commonly Used Isotopes 32Phosphorous 1–3 weeks 33Phosphorous 4–12 weeks 35Sulfur 2–6 weeks 125Iodine 3–12 weeks 3 Hydrogen 1–12 months 14Carbon 1–2 years